The present invention relates to a method for producing nucleic acid polymers.
The increasing use of recombinant genes in genetic engineering and biotechnology and in the medical analytical field has created a great demand for methods of the xe2x80x9cde novoxe2x80x9d synthesis of long nucleic acid chains. In many cases the synthetic production of arbitrarily chosen nucleic acid sequences can be a time-saving alternative to troublesome cloning and modifying methods. A routine synthesis of long nucleic acid chains can also offer decisive advantages in the case of xe2x80x9cdrug designxe2x80x9d, e.g. in the production of xe2x80x9ccustom-madexe2x80x9d antibodies, inhibitor/activator molecules, ribozymes, or in DNA chip technology. Furthermore, purposefully modified nucleotides, e.g. labeled by (fluorescent) dyes or enzymes, could be incorporated into a nucleic acid chain in this way. Apart from the examples given here, there are many further possible applications for purposefully produced nucleic acid polymers.
In the simplest variant of conventional gene synthesis, i.e. direct cloning, two long oligonucleotides which are fully complementary to each other are hybridized with one another. On account of the present technical limitation in the production of oligonucleotides to a length of 150 to 200 bases, the size of the resultant hybridization products is also limited to such a range of length.
A further method for producing long nucleic acid polymers is the so-called fill-in method in which two single-stranded nucleic acid chains are hybridized with each other, and protruding ends are filled with the help of DNA polymerases so that the double-stranded product is longer than the oligonucleotides used. However, even with the fill-in method, it is not possible to produce a product that is longer than the sum of the nucleic acid chains used.
In the xe2x80x9cshot-gun gene synthesisxe2x80x9d complementary single-stranded oligonucleotides are directly transfected into cells together with an expression vector which has been opened by restriction enzymes, and a circularized product can here only be obtained if all of the partial sequences are ligated by the enzyme machinery of the host organism in a suitable way. The efficiency of successful ligations in this method is in general very limited, in particular when many oligonucleotides are used for gene synthesis.
In 1972 Khorana developed a method named after him, in which several chemically synthetized oligonucleotides of an average length of 15 nucleotides, which in a suitable arrangement are overlapping without any gaps, were enzymatically joined by polynucleotide ligase to obtain a double strand (Khorana, H. G. et al., J. Mol. Biol. 27, 209-17, 1972, and follow-up publications; Khorana, H. G. et al., J. Biol. Chem. 251, 3(10), 565-70, 1976 and follow-up publications). Sequential ligation of a few (4-8) oligonucleotides for obtaining longer intermediate products, purification of the intermediate products and subsequent ligation of the intermediate products with one another resulted in the synthesis of double-stranded nucleic acid chains with a length of 514 base pairs (Edge, M. D. et al., Nature 292, 756-62, 1981), later with a length of up to about 1000 bp, which could subsequently be cloned in bacterial expression vectors.
These methods have several decisive drawbacks:
1.) After each ligation step the products or intermediate products had to be purified by separation on a polyacrylamide gel (PAA gel) to eliminate undesired by-products of the ligation reaction. Such a time-intensive working step requires great efforts with respect to personnel and costs.
2.) During elution of the intermediate and final products from the PAA gel considerable losses in yield had to be accepted.
3.) The so far longest product that could be produced with the help of said technique had a length of about 1000 base pairs. Since most of the eukaryotic and prokaryotic genes have a coding sequence of 300 to 3000 base pairs on the average, such a length is not sufficient for most applications.
4.) For the necessary purification via PAA gels the nucleotides were normally radioactively labeled in the method described by Khorana to be able to identify the bands of the desired products in the gel. The use of highly radioactive 32P labels constitutes a potential risk which could not be avoided in said method.
It is therefore the object of the present invention to provide an uncomplicated, reliable and inexpensive method for the synthesis of nucleic acid polymers of a length of more than 1000 bases in one step, wherein the above-mentioned drawbacks can be overcome.
Said object is achieved by a method for producing a nucleic acid polymer, comprising the following steps:
a) providing 2 or more linkable oligonucleotides which in a continuous arrangement and after linkage can form a primary strand, and one or more non-linkable oligonucleotides, each of the non-linkable oligonucleotides comprising two adjoining regions the first of which is complementary to the 3xe2x80x2 end of a linkable oligonucleotide and the second of which is complementary to the 5xe2x80x2 end of a further linkable oligonucleotide,
b) hybridizing oligonucleotides for the primary strand with the complementary regions of the non-linkable oligonucleotides, and
c) linking the oligonucleotides of the primary strand.
The method according to the invention is schematically shown in FIGS. 1 and 2. In contrast to the Khorana method, it offers the decisive advantage that the synthesis of a single-stranded nucleic acid polymer can be carried out in a single reaction batch. All of the linkable and non-linkable oligonucleotides that are required for the synthesis of the primary strand are here used at the same time; the addition of a linker yields a primary strand of covalently linked oligonucleotides which can reach a length of more than 1000, e.g. 1500, bases.
In preferred embodiments steps (b) and (c) are repeated several times, and the oligonucleotides which were previously hybridized with one another are separated from each other prior to each repetition of said two steps, i.e. the double strand previously formed by hybridization is denatured. Denaturation can be carried out by increasing the temperature or by increasing the pH in the manner known to one skilled in the art. The repeated denaturation and renaturation with subsequent linking considerably improves the yield in primary strand which, otherwise, is e.g. impaired by chain terminations which are the result of the incorporation of incompletely phosphorylated primary strand oligonucleotides. The influence of such chain terminations on the total yield is minimized by repeating steps (b) and (c).
According to the invention steps (b) and (c) are carried out not only once, but several times. In a preferred embodiment, they are repeated 1 to 8 times, particularly preferably 3 to 5 times.
The number of the oligonucleotides used can vary between 2 linkable oligonucleotides and 150 linkable oligonucleotides. The number of the non-linkable oligonucleotides is always equal to the number of the linkable oligonucleotides, by one higher or by one lower, i.e. at least one. Between 5 and 100 linkable oligonucleotides are preferably used; particularly preferably between 10 and 50 linkable oligonucleotides.
The linking of the linkable oligonucleotides of the primary strand may comprise various reactions. In this instance linking means e.g. a reaction of an enzymatic, chemical or also photochemical kind. For instance in the case of enzymatic linking (ligation), T4 DNA ligase is e.g. used as the linker. In the preferred embodiment, use is made of a thermostable ligase, e.g. Pfu ligase (Stratagene) which offers the advantage that in the case of repeated cycles of denaturation by way of temperature increase, hybridization and ligation of the oligonucleotides, no new enzyme has to be added whereas this is the case when thermolabile DNA ligases, such as T4 DNA ligase, are used.
Furthermore, the use of said thermostable ligase makes it possible to carry out the steps of hybridizing and ligating the oligonucleotides at high temperatures of 45xc2x0 C. to 80xc2x0 C., preferably 70xc2x0 C. Such stringent temperature conditions mean that the step of hybridizing the oligonucleotides is carried out in a complementary arrangement with a high specifity and that the amount of by-products obtained is considerably smaller than in the Khorana method. Ligations with conventional ligase are normally carried out within a temperature range of 40xc2x0 C. to 4xc2x0 C. and can thus lead to a higher amount of unspecific hybridizations by mispairing or secondary structures.
A chemical linkage of oligonucleotides, which has been described by Goeddel and colleagues (Goeddel, D. V. et al., PNAS 76, 1979), is also possible.
A further possibility of linking oligonucleotides with one another is photochemical linkage. In this process the oligonucleotides to be linked must be labeled at the terminal nucleotides with photosensitive molecules which contain carbon-carbon double bonds and may be subject to a (2+2) photocyclodimerization, e.g. stilbene, allene, mono-, di- and triene dicarboxylic acid derivatives or styrene derivatives.
According to the invention the non-linkable oligonucleotides just serve as a template for forming the nucleic acid polymer. In a preferred embodiment said oligonucleotides are not phosphorylated at their 5xe2x80x2 end. As a result, an enzymatic linkage of said oligonucleotides, which are only responsible for the annealing of the linkable oligonucleotides in the desired arrangement, is not possible.
Non-linkable oligonucleotides can also be used in which e.g. the 3xe2x80x2 end is a di-deoxynucleotide or a thionucleotide. Corresponding modifications are also possible at the 5xe2x80x2 end. Furthermore, the method can be carried out with non-linkable oligonucleotides which are phosphorylated at the 3xe2x80x2 end. Such a modification also prevents the enzymatic linkage of the oligonucleotides. Whenever the method of the invention is not carried out by means of enzymatic ligation, but is performed by way of chemical or photochemical linkage, the non-linkable oligonucleotides must be conceived accordingly in such a manner that under the conditions chosen for linking the olignucleotides forming the primary strand, they cannot form a covalent bond with the respectively adjacent non-linkable oligonucleotide.
The oligonucleotides used in the method of the invention may have a length of 30 to 1500 nucleotides. The length of the respectively selected nucleotide depends on several factors, including the probability of forming secondary structures and the purity of the selected start materials. However, it is preferred that the oligonucleotides of the primary strand have a length of 30 to 200, particularly preferably, 30 to 100 or 30 to 60 nucleotides.
Single-stranded nucleic acid polymers already produced during synthesis can be combined in a further step, again with the method of the invention, to obtain single-stranded nucleic acid polymers having a length of several kilobases (FIG. 3). To this end, the method is carried out as described above, wherein nucleic acid polymers of e.g. 1500 bases can be used as linkable olignucleotides for the primary strand.
The non-linkable oligonucleotides which are to ensure a correct arrangement of the oligonucleotides of the primary strand side by side may optionally have a length of only 30 to 50 nucleotides. This order of magnitude permits hybridization with two oligonucleotides of the primary strand that are to be arranged side by side. Non-linkable oligonucleotides that cover the whole primary strand need not be provided for. However, the use of non-linkable oligonucleotides with up to 300 nucleotides or more is of course also possible and can certainly make sense in cases where strong secondary structures would otherwise be formed, for instance, in the primary strand.
Each of the non-linkable oligonucleotides contains two adjoining regions which exhibit complementarity to the 3xe2x80x2 end and 5xe2x80x2 end, respectively, of two adjacent oligonucleotides for the primary strand. The regions of complementarity between the linkable oligonucleotides for the primary strand and the non-linkable oligonucleotides of the complementary strand have each a length of about 15 to 30 base pairs, preferably 20 to 25 bp (see FIG. 2).
A preferred embodiment of the method consists in that in a preceding reaction the terminal oligonucleotides for the primary strand and/or oligonucleotides which are fully or partly complementary to the terminal oligonucleotides of the primary strand are annealed to the ends of a linearized vector, that they are linked with the vector and that subsequently the method set forth in claim 1 is carried out. The vector ends are here preferably cohesive. This method yields a circularized product which can directly be transfected into a suitable host organism, e.g. bacteria, mammal or insect cells. xe2x80x9cTransfectionxe2x80x9d in this instance means various techniques, such a electroporation, microinjection, infection, transfection supported, for instance, by xe2x80x9cCaxe2x80x94PO4xe2x80x9d, DEAE, hydrophobic molecules, etc.
In a preferred embodiment, either the 5xe2x80x2 terminal oligonucleotide of the primary strand at its 5xe2x80x2 end or the 3xe2x80x2 terminal oligonucleotide of the primary strand at its 3xe2x80x2 end is provided with a hapten, or both terminal oligonucleotides are provided with different haptens. This permits a fixation of the hapten-coupled oligonucleotide to a fixed carrier either before or after its linkage to further oligonucleotides of the primary strand. This allows a separation of the hapten-coupled strand from all precursors and by-products without hapten, e.g. under denatured conditions (e.g. pH 13). The respectively selected hapten-carrier combination must be stable under the conditions set. A preferred hapten is e.g. biotin which is bound by strepavidin coupled to a carrier. Furthermore, the oligonucleotides can be coupled with antigens which are recognized and bound by antibodies fixed to a carrier. The further synthesis of the primary strand or one of the further steps described in the following can then be carried out on said carrier. The second hapten could e.g. be useful for detection methods to be carried out with the nucleic acid polymer at a later time.
In a further preferred embodiment a polymerase enzyme is added to the reaction batch of a synthesis reaction carried out according to the present method to form a nucleic-acid double strand from the primary strand, which is a nucleic-acid single strand, or to amplify the complementary strand (i.e. the counter strand to the primary strand) in a purposeful manner (asymmetrical PCR). An oligonucleotide which is complementary to the 3xe2x80x2 end of the complete primary strand (reverse primer) serves as the specific primer.
In a preferred embodiment the reaction batch of a synthesis reaction performed according to the present method has added thereto a polymerase enzyme and, in addition, terminal forward and reverse primers in excessive amounts with respect to the synthesis product used. Double-stranded nucleic acid, polymers are thereby formed from the single-stranded nucleic acid polymers under the principle of the polymerase chain reaction (Saiki et al., Science 239, 487-491, 1988).
The polymerase enzyme is here selected from the group of the DNA polymerases, e.g. E. coli polymerase I, Klenow polymerase, T4 DNA polymerase and reverse transcriptase. The nucleic-acid double strands formed thereby may either be DNA-DNA double strands or DNA-RNA double strands (Kleppe et al., Proc., Natl. Acad. Sci. 67, 68-79,1970).
In a particularly preferred embodiment the added polymerase is a temperature-stable polymerase, e.g. Taq polymerase. Even after several temperature cycles the temperature-stable enzyme will hardly lose any of its activity and can therefore catalyze the polymerase reaction of several successive cycles.
The polymerase reaction is preferably repeated several times to exponentially amplify the nucleic-acid double strand. In accordance with the principle regarding polymerase chain reaction each cycle comprises a denaturation of already existing nucleic-acid double strands by taking standard measures, e.g. by increasing the temperature or the pH, annealing the terminal primer under suitable conditions or a polymerase-catalyzed nucleic acid synthesis. In the case of a temperature-stable polymerase the addition of further polymerase is not necessary; however, in cases where one of the polymerases that are not temperature-stable has been used for the polymerization reaction, said polymerase is inactivated by thermal denaturation; in such a case fresh polymerase enzyme has to be added in each cycle. Normally, the polymerase reaction is carried out 5 to 15 times, preferably about 8 to 12 times. The essential advantage offered by repeating the polymerase reaction is that on account of the position of the primers (terminal), purposefully complete synthesis product can be amplified in an exponential manner.
The double strands are normally denatured at temperatures of more than 90xc2x0 C. The reaction batch is then slowly cooled so that, depending on their composition, the terminal primers can hybridizexe2x80x94at temperatures ranging from 80xc2x0 C. to 45xc2x0 C.xe2x80x94with the single strands that will then be present. The polymerase-catalyzed DNA synthesis can be carried out using a temperature-stable enzyme, whereby the formation of unspecific hybrids is minimized.
The subject matter of the present invention is further a single-stranded nucleic acid polymer which has been obtained with the method according to the invention. By skillfully coupling the method of the invention (by which single-stranded primer strands are first produced) with the fill-in method, it is easily possible to produce double-stranded nucleic acid polymers of several 1000 bases (FIG. 4). To this end the primary strands, for instance, have to be conceived such that they are complementary to one another at their 3xe2x80x2 ends within a range of a few base pairs, e.g. 20 to 60, preferably 30 to 40 base pairs, so that in a polymerase reaction subsequently taking place in vitro or in vivo the 3xe2x80x2 end of each primary strand can serve as a primer for a template-dependent polymerase.